Production of a protein delivery system for in vivo therapeutic treatment

ABSTRACT

Disclosed is a generalized protein delivery system that (i) contain one or more recombinant molecule(s) that are expressed on a cellular surface and (ii) incorporated and/or associated with a particle that does not require cellular entry for delivery. The recombinant protein is preferably a protein, peptide, and/or antibody. The particle is preferably a virus-like-particle devoid of any intrinsic infectivity. Also disclosed is a method for formation and production of the particles able to carry at least one recombinant molecule. In addition, methods are disclosed for the delivery of the recombinant particles into a mammalian host for the prophylactic therapeutic treatment and/or as a diagnostic reagent for human diseases.

FIELD OF THE INVENTION

This invention relates to the field of delivery of therapeutic proteinsfor prophylactic purposes in mammals by the production of a proteindelivery system capable of delivery of recombinant proteins, stimulationof biological processes, inactivation of inhibitory chemical and/orbiological factors, and scavenging and/or detoxifying any molecule fortherapeutic benefit either in vitro or in vivo.

BACKGROUND OF THE INVENTION

Eukaryotic protein expression systems are frequently employed for theproduction of recombinant proteins as therapeutics as well as researchtools. Most commonly used expression systems are based on stablytransfected Chinese hamster ovary (CHO) cells and infection of insectcells by recombinant baculoviruses. Although much success has beenassociated with protein production in established protein expressionsystems, numerous obstacles are the source of structural heterogeneitythat could lead to functional deficiencies. Comparison of intracellulartransport and processing of a recombinant glycoprotein in these systemsdiffer considerably among each other and often different from the nativeprotein. Intracellular proteins in both systems are associated witholigosaccharide protein glycosylation, but are absent orunder-represented in sialylated glycoforms, in addition to post-proteinglycan processing. Differential post-translational modification ofsynthesized proteins can profoundly affect three-dimensional structureand biological function.

Sialylation of N-glycans associated with human serum proteins has acentral role in determining its circulatory clearance rate. Higher thesialylation, the longer the clearance rate in the mammalian circulation.Clearance times can change from minutes to hours with increased proteinsialylation. Although the degree of sialylation may play an importantadaptive response during early development, the reduced sialylationduring production of recombinant proteins that require sialylation inour present eukaryotic expression systems has made these recombinantproducts almost useless as therapeutics. Approaches to overcome theseinnate deficiencies have either exposed recombinant proteins in vitro toexoglycosidases and sialyltransferase or have introduced liver derivedbeta-galactoside alpha-2,6-sialyltransferase cDNA by gene transfer intocells producing the recombinant protein. The in vitro incorporation ofsialic acid into neuraminidase-treated recombinant proteins (developedspecifically to allow efficient sialic acid capping ofbeta-galactose-exposed termini) has been shown to saturate >70% of thetheoretical acceptor sites. Similarly, recombinant proteins produced bythe gene-modified cells displayed a significantly higher proportion offully sialylated glycans. Both sialylated recombinant serum proteinsmade from genetically modified cells and those subjected to extensivesialylation in vitro exhibited increased circulatory retention timesapproaching the degree of sialylation and retention times found onproteins purified from native tissue sources. Although these approachesaccomplish the desired result, they are cumbersome and time consuming.They are costly and yields are low. Liver cells are the in vivo sourceof many of these highly sialylated glycoproteins containingsialyltransferase that are involved in the sialylation ofO-glycosidically linked carbohydrate chains on serum glycoproteins. Thisobservation suggests that hepatic cells rather than Chinese hamsterovary cells or baculovirus would be the cell line of choice whenexpressing these serum proteins. Thus, it can be concluded thatexpression of these recombinant proteins from the same cells thatproduce these proteins in vivo may improve their pharmacokineticbehavior and their prophylactic therapeutic usefulness either in vitroor in vivo.

In addition to sialylation and similar post-translational proteinmodifications, another innate deficiency in our present eukaryoticprotein expression systems is the ability to multimerize monomeric formsof expressed recombinant proteins. In most if not all cases, monomers ofexpressed proteins require higher forms of structure in order to carrierout their intended function. Multimerization of protein monomers intodimeric, trimeric, or tetrameric structures rely on protein-proteininteractions and in some cases disulfide-linkage. In some cases theseinteractions are intrinsic to the molecule; in others, cellular encodedproteins facilitate these oligomeric super-structures. There is anincreasing awareness from a spectrum of genetic deficiencies thatmutations in a “linking or anchoring” protein and not in the specificgene itself is responsible for various congenital syndromes. Thesedeficiencies are caused by an uncoordinated expression of proteinsubunits and linking/anchoring proteins that normally determines thepattern of molecular forms, which in turn determines the localizationand functionality of the resulting protein.

Thus, our present state of the art for eukaryotic protein expressionsystems fail in at least two post-translational modifications that arerequired for the high level of expression of recombinant glycoproteinswhose sialic acid content is important for their function andpharmacokinetic behavior—the level and nature of N-glycan capping andsubunit assembly.

As the state of the art in molecular recognition of biowarfare agentsand other pathogens improves, there is an increasing need to developquick acting and efficient therapeutic recombinant bioscavengers fordefense against these chemical and biological agents. The biocatalyticdestruction of organophosphates has become an important focus area andefficient technologies are being sought for counteracting chemicalweapons to afford protection against nerve agents and pesticidepoisoning. Novel methods have been advanced using enzymes hydrolyzingorganophosphates as potential catalytic scavengers againstorganophosphate poisoning such as organophosphorous hydrolase fromPseudomonas diminuta, carboxylesterase, and the role ofphosphotriesterases in the detoxification of organophosphorus compounds.With the recognition that broad-spectrum organophosphorus insecticidewere designed to produce acute cholinergic effects by inhibition ofacetylcholinesterase and the knowledge that organophosphorus insecticideexposure exhibits only moderate acute toxicity in mammalian species dueto rapid detoxification of the active metabolite byacetylcholinesterase. Research has focused on the use of nativecholinesterases as a mode of treatment to prevent organophosphatetoxicity. Research in this area demonstrated the ability of nativecholinesterases as an effective mode of pretreatment to preventorganophosphate toxicity in mice and rhesus monkeys. Tissue-derivedcholinesterases were compared to recombinant cholinesterases afterintravenous injection into mice and rhesus monkeys, illustrating thereduced circulatory half-life of the recombinant protein. The summary ofa large body of research points to the innate deficiencies insialylation and monomers assembly in tetrameric active protein subunitsin our present eukaryotic expression systems as the underlining reasonsfor the observed instability in vivo. Until these obstacles areovercome, recombinant approaches to therapeutic detoxification ofchemical and biological agents will not be a realistic preventionstrategy for preventing toxicity. The current, non-recombinant, approachof isolating cholinesterases from natural sources of human plasma andother blood by-products is impractical due to the large quantitiesrequired and the potential biohazard associated with the presence ofknown (HIV-1, hepatitis, prions, etc.) and yet to be discoveredpotential pathogen present in pooled human-derived tissue products.Recombinant molecular technology is the only practical way to accomplishthe goal to attain large quantities of human cholinesterases. We needchemical/biological defense agents displaying a high stability uponlong-term storage in order to define its therapeutic capacity in vivo,its pharmaceutical properties for clinical trials, and if the resultsare successful, for general use in detoxification of organic compounds.Thus, there is a requirement for a generic technology that hasversatility and economy-of-scale that would allow production andpurification of kilogram quantities of any number of specificbioscavengering agents.

In addition to bioscavenging agents, the above discussion relates to anyand all in vitro synthesized amino acid containing product. Recognitionof proteins, peptides, and antibodies by their biological cellularreceptor is dependent on the “decoration” of the primary amino acidsequence with sugars, lipids, and nucleic acids modifications. Thesemodifications that effect biological processes, which are mostlypost-translational, are performed within specialized cells through outthe mammalian body independent of the molecule being eukaryotic orprokaryotic in origin. The biological processes include cellular andnon-cellular, but molecular receptor-mediated binding of an amino acidcontaining sequence to induce or inhibit differentiation, to stimulateor suppress immune responses, to attract or repel cells and/orbiomolecules, to prevent organ and/or host toxicity—basically toinfluence biological processes in vitro and/or in vivo in a positive wayto achieve a favorable therapeutic or preventive outcome in an mammalianhost, especially when that mammalian host is human.

Many viruses produce degenerative changes in cells when replicating insusceptible cells in culture in vitro. These characteristic changes arecalled cytopathic effects and are associated with certain morphologicchanges in the host cell. The intracellular sites where the events ofviral replication take place vary among the viral families. Envelopedviruses mature by a budding process, although some budding occurs withnon-envelope containing viruses. For envelope viruses, viral-specificenvelope glycoproteins are inserted into cellular membranes and theviral nucleocapsids then bud through the membrane at these modifiedsites. In this process, the virus acquires their envelope forinfectivity and can also acquire cellular-related molecules. Studieswith HIV, Influenza, and Chlamydia have shown viral particles that haveincorporated HLA molecules into the mature virus particle. During theinfection the cell is destroyed and the virus particles are releasedinto the culture supernatant. The amount of infectious virus present inthe cell culture fluid can be titrated and infectivity inactivated by avariety of methods. Although inactivated virus particles have lost theability to replicate, they maintain their structure and as detailed inthis application, they can be used as a scaffold to carry cell surfaceexpressed molecules.

The final step in the lytic cycle of enveloped viruses involves thebudding of the newly formed particles from cellular membranes. Studiesof viruses that obtain their envelope from the plasma membrane haveestablished the dependence of virus budding on interactions with viralproteins. For the alphavirus—Semliki Forest virus, it has beenestablished that virus budding is strictly dependent on interactionsbetween the transmembrane spike protein and the internal nucleocapsid.In retroviruses, however, interactions between the cytoplasmic tail ofexternal viral protein and the internal viral components are not aprerequisite for virus budding since expression of the gag protein aloneis sufficient to drive budding of virus-like-particles. A differentmechanism directs the assembly and release of coronavirus particles. Theparticles assemble at intracellular membranes and expression of viralmembrane proteins drives the assembly and budding ofvirus-like-particles. Overall matrix protein plays a pivotal role inassembly of RNA viruses. The M1 proteins of vesicular stomatitis virus,human parainfluenza virus type 1, and influenza-A has intrinsic buddingactivity when expressed alone. In fact, similar to retroviruses,interactions between the internal viral component and the cytoplasmictail of external virus proteins are not an absolute requirement forvirus particle formation. Similar to inactivated virus particles,virus-like-particles maintain their structure and as detailed in thisapplication, they can be used as a scaffold to carry cell surfaceexpressed molecules.

SUMMARY OF THE INVENTION

This invention provides for the formation, production, and in vivodelivery of recombinant molecules for therapeutic and diagnosticpurposes. The invention could contain one or more than one molecule orcontain native cell surface components from a particular cell type.Molecules preferably include any amino acid moiety-containing molecule,but other molecules captured during the process covered by thisinvention could be envisioned. Formation of specific molecules could beengineered genetically by molecular biology techniques to be expressedon the surface of cells that alone or together with native molecules onthe said cells' surface, forms the essence of the invention. Theformation and production of the invention involves the removal of cellsurface membrane components as a consequence to the budding of particlesfrom within the cell. The particles could be made of single or multiplecomponents. Components are envisioned to be viral in origin, but couldbe induced by non-viral methods, or natural to the cell selected host.The invention is preferably for in vivo delivery, but could be used invitro for induction or maintenance of cellular processes. Processesinclude, but not limited to, cellular signaling, cellular induction,cellular suppression, cellular attractant, cellular differentiationand/or cellular or molecular scavenging. In vivo delivery could be byintravenous injection, but other routes include but are not limited tooral, suppository, intramuscular, inter-cranial, inter-peritoneal, ordirectly into mammalian organs, capillaries, ducts, or lymphoid systemeither alone or associated with biological or non-biological materialsor devices. Inter-respiratory devices, cutaneous and topicalapplications are also envisioned within this invention. In addition theapplication of the invention as aerosols, creams, puffers, or onsurfaces, is included in this invention. Surfaces include, but notlimited to, synthetic, non-synthetic, biological, or non-biologicalmatrixes including autologous, allogeneic, and xenogeneic extracellularmatrix materials. Therapeutic purposes encompass all procedures and/orprocesses that result in the improvement or intended improvement of thehealth and well being of an inflicted human or mammalian host.

In addition to therapeutic protein production, this inventionencompasses counter-measures against chemical and biological defenseagents using recombinant molecular technology could be envisioned, wherethe counter-measure is a biological or chemical molecule(s). Thecounter-measure is preferably a molecule that binds and inactivates achemical or biological toxin, or inhibits cellular entry of said toxinor biological agent, thereby preventing damage to biological human andmammalian tissues. Although the counter-measure is conceived to be aprotein expressed from a eukaryotic cell or protein expression system bya defined nucleic acid sequence(s), it need not be limited to suchbiological molecules and as a protein, it may require additionalpost-translational modifications to enable the counter-measure toprovide the necessary disabling function(s). The invention is intendedfor in vivo use in any recipient (human or mammalian) requiringdetoxification, although in vitro usages can also be envisioned.

In addition, this invention relates to the prevention of toxicity and/ororgan failure do to the accumulation of metabolites and/or deposits dueto the nature of the recombinant protein being synthesized from cellularsources foreign to the native molecule and host. In this capacity, theinvention is envisioned to produce a biological carrier delivery systemwhere the captured molecule(s) is indistinguishable from the nativemolecule(s) with the exception of being embedded or attached to abiological carrier (inactivated virus or virus-like-particle). Themolecule or molecules would be in the native configuration containingall possibly required modifications that are native to said molecule(s),and as such, contains the innate ability to behave and maintain the samebiologically activity as if the molecule(s) were synthesized in vivo. Assuch the molecule would be native to the host and as such will not causethe accumulation of metabolites that would be toxic to the host, norresult in deposits in organs that could be detrimental to the host.Toxicities and unusual deposits are sometime intrinsic to recombinantmaterial synthesized in either commonly used in vitro systems,especially when these molecules are synthesized in non-mammalian systemslike yeast or bacteria.

In one aspect the invention is a bio-scavenger, a receptor or an enzyme.As a bio-scavenger, the invention could bind, chelate, sequestered,and/or inactivate a chemical or biological agent. As a receptorexpressed on the surface of a human or mammalian cell that inducesparticle formation by budding off membrane pieces containing therecombinant receptor, the invention provides a mechanism to bind,chelate, sequester, and potentially clear any biological inhibitoragent(s), molecule(s), or process from a human or mammalian biologicalbody or system. As a receptor, the molecule may need to be modified tomake the molecule cell surface expressed-for example addition, removal,and/or replacement of signal peptide, intracellular, transmembraneand/or other molecular domain sequences. As a said receptor, the agentbinds, sequesters, and clears the biological inhibitor molecule or toxinas a complex from the body. As an enzyme the agent binds, inactivates byenzymatic cleavage or non-enzymatic hydrolyze to metabolites that are nolonger harmful to human or mammalian tissues and/or hasten the removalof the toxin from the host. Inactivation can occur, but not limited toenzymatic cleavage, blocking of reactive moieties, masking of activesite(s), sequestering to certain tissues, and/or clearance of the toxinas a bound or unbound complex. In one embodiment of this aspect, thecDNA for a specific biological molecule or molecules is introduced bymechanical, physical, chemical, or viral means into cells capable ofhigh-level cell-surface protein expression. Mechanical, physical, andchemical means include but not limited to electroporation, and/orlipid-mediated, polyethylene glycol, Sendai virus membrane fusion thatbypasses the cellular membrane to gain access to the cellular chromatinstructure where integration may or may not occur. Viral mediateddelivery mechanisms include but not limited to murine leukemia virus(MuLV), adenovirus, adeno-associated virus (AAV), lentivirus, andcanarypox vectors. The human or mammalian cells capable of high-levelcell-surface protein expression could be any primary, transformed,and/or established cell line of autologous, allogeneic, or xenogeneicnature.

In another aspect the invention provides co-expression of moleculesand/or portions of molecules that either facilitates assembly,configuration, conformation, or co-expression of proteins to stimulateone or more than one cellular process at a time. These modifications orco-expressed molecules assist in the final biological activity of theexpressed recombinant proteins are covered within the scope of thepresent invention. Biological activity can be, but not limited to: (i)binding affinity/avidity of inhibitor molecules; (ii) enhancement ofenzymatic hydrolyze; (iii) blocking of the inhibitor molecule mode ofaction; (iv) stabilizing the recombinant protein itself and/or when in arecombinant protein: inhibitor molecular complex; (v) enablingbiological activity over extended periods of time in biological fluids;(vi) enhancing clearance and/or removal from the host once therecombinant protein is complex with the inhibitor molecules; (vii)delivery of any molecule(s) for preventative, therapeutic, and/ormaintenance purposes; and/or (viii) activation, induction,differentiation, homing and/or attracting cells in vitro and/or in vivo.These biological activity properties could be achieved by, but notlimited to: (i) the introduction of a genetic sequences into a cell;(ii) expression of a protein(s) on the cell surface; (iii) expression ofmolecule(s) that enhance the biological effect; (iv) production ofparticle release either innate to or induced by the introduction ofviral or non-viral components by either mechanical, chemical, and/orviral vector means; (v) harvesting of released particles; (vi)concentration of released particles; (vii) inactivation of releaseparticles; (viii) lyophilization of particles for long-term storage;(ix) exposure of cells, organs, or biological systems in vitro toparticle preparation; and/or (x) delivery of particle preparation invivo by any conceivable route.

In another aspect the invention provides for molecules containing theappropriate post-translational modifications required for human andmammalian proteins found in vivo. This is due to the de novo synthesisof protein molecule(s) within the same cells that the protein isnaturally expressed in situ. The invention supplies advantages to therecombinant molecule(s) in that the formation and production of the saidmolecule is in accordance with, and as close as possible to thenaturally expressed molecule. The adherence to the native protein inconformation, configuration, multimerization, and post-translationalmodifications that include but not limited to glycosylation,polyADPribosylation, and myristylation, improves the functionalparameters associated with the said molecule(s). The gene and/or nucleicacid sequence would be either naturally expressed on the surface of thecell or genetically modified by the addition of heterologous sequencesto the said sequence in such a way that the said molecule would be cellsurface expressed. Cell modification could be by gene transfer. Anynumber of viral or non-viral vectors or direct delivery methods couldintroduce any number of genes to express proteins onto the cells'surface. The cell surface expressed protein(s) are captured intoparticles as the particles are released from the cell. Genes could codefor molecules, including but not limited to, enzymes, receptors,receptor ligands, cytokines, growth factors, and antibodies. Inaddition, the introduced gene(s) could code, but not limited tomolecules involved in oocyte and/or cellular differentiation,homeostatic maintenance, and/or initiating intracellular signalingpathways that either enhance or inhibit biological processes.

The present invention describes a protein delivery technology that hasdemonstrated the ability to incorporate over-expressed proteins intoeither active or inactive viral particles and/or virus-like-particles,but the invention is not limited to viral induced budding components.The process relies on the biological process of particle release toremove pieces of the cellular membrane while exiting a said host cell.Stable cell lines are loaded with a recombinant protein of interest onthe surface of cells by standard molecular biological transfection ortransduction techniques. The modified cell may be either (i) infectedwith a virus that is able to productively induce a lytic or non-lyticinfection resulting in virus progeny; (ii) chronically infected andcontinuously release virus particles; (iii) transfected or transducedwith one or more viral component either transiently or permanentlyexpressed that results in particle release; and/or (iv) naturally orartificially induced to release particles of cellular origins capable ofcapturing molecules expressed at cellular membranes. In all cases, thereleased particles contain the same over-expressed protein present onthe host cells' surface and as such serve as a novel delivery system forrecombinant molecules. In this way, single or multiple molecules areexpressed with similar native structure to the naturally expressed humanor mammalian protein.

The “capture” of a protein on the surface of a particle simplifies theprocess of synthesizing and purifying recombinant molecules and/orproteins to harvesting virus particles. Thus, in vitro recombinantprotein systems can be simplified to purification ofviral/non-viral/cellular particles or viral-like-particles usingstandard generic techniques. At the same time this technology insuresproper orientation, conformation, and post-translational modificationsof the synthesized protein, since the protein is made de novo. Thesemodifications are not being met for recombinant proteins expressed instandard systems presently in use—like Chinese hamster ovary cells (CHO)and infection of insect cells by recombinant baculoviruses due tointrinsic deficiencies in these systems. To further insure properpost-translational and conformational recombinant protein synthesis,host cells that naturally express the protein could be chosen as thehost for in vitro synthesis.

In summary, the present invention describes the utility of a process todeliver and produce recombinant molecules for therapeutic and diagnosticpurposes. For therapeutic purposes, the invention is a protein deliverysystem; for diagnostic purposes, the invention is a method to capturenative molecules representing innate structures identical to those foundin vivo for measurement of immune responses or as a source of nativemolecules for purification and diagnostic kit purposes, including butnot limited to molecules from hazardous bio-organism. The invention hasa wide range of applications, including but not limited tocounter-measures against chemical and biological defense agents. Theinvention provides a method to synthesize, produce, and purifybiologically active molecules incorporated in and/or attached to intactparticles for therapeutic, diagnostic, reagent and/or protectivepurposes. The goal of this invention is to bestow biological propertiessimilar to or improved upon that found in natural biological systems.The invention is a biological agent expressed as a particle containingone or more recombinant protein for in vitro and in vivo use to modify,enhance, protect, and/or induce cellular processes.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described by the accompanying drawings and thedescription thereof herein, although neither is a limitation of thescope of the invention.

FIG. 1 is a schematic representation of the protein delivery systemcovered by the present invention. The figure illustrates theintroduction of nucleic acid sequences into a specific host cell and theexpression of the nucleic acid molecules onto the cell surface of saidhost cell. In this embodiment, the introduced transcribed and translatednucleic acid sequences expressed on the host cell surface are “captured”upon release of the infectious agent that is used to infect the nucleicacid modified host cell. The figure further illustrates the harvesting,concentration, and inactivation of the infectious agent releasedparticle preparation. In this figure, the protein delivery system isreferred to as “Biological Carriers.” The term biological carriers werecoined to emphasis both the biological nature of the technology andtheir ability to carry fully expressed molecules.

FIG. 2 is a schematic representation of one potential application of the“biological carrier” protein delivery technology—its ability to interactwith T-cells to influence the activation and formation of immune cellsdirected against specific forms of cancer and/or infectious diseases.The diagram shows the particles interacting with T-cells, but theseinteractions could be with any cell type in vitro or in vivo. Theactivation is envisioned to be by receptor-mediated signal transductioninduced pathways, but mechanisms presently known or unknown may beinvolved. In this figure, the protein delivery system is referred to as“Biological Carriers.” The term biological carriers were coined toemphasis both the biological nature of the technology and their abilityto carry fully expressed molecules.

FIG. 3 is a schematic representation of the use of this protein deliverysystem with respect to both an Acquired Immunodeficiency Syndrome (AIDS)therapeutic and diagnostic reagent. The figure illustrates the formationof particles from a chronically infected cell line where the infectiousagent is continuously budding from the cell lines' surface. In thepresent diagram, the human immunodeficiency virus (HIV), the etiologicalagent of AIDS is illustrated, but similar cell lines harboringinfectious agents or portions of infectious agents could be envisioned.As an AIDS therapeutic, costimulatory molecules are introduced into thechronically-infected cell line, which together with HIV-specificantigens processed as peptides in major histocompatibility (MHC)molecules that are presence on the cell line due to chronic expressionof HIV, should be able to stimulate immune responses when contacting theappropriate cells either in vitro or in vivo. As an AIDS diagnostic, theparticles could be used as a preparation for the purification ofspecific viral proteins and/or nucleic acids, in addition to an antigenpreparation for detection of immune responses against HIV in humans foranalysis of the presence of antibodies against specific viral antigens.Detection could be by ELISA, PCR or Western Blot, but not limited tosuch detection systems. In addition, the schematic illustrates the“capturing” of cellular surface molecules that are native to said hostcell, are also present in the final particle preparation. In thisfigure, the particles are identified as BC, which refer to BiologicalCarriers. The term biological carriers were coined to emphasis both thebiological nature of the technology and their ability to carry fullyexpressed molecules.

FIG. 4 is a schematic representation for using virus-like-particles(instead of infectious virus particles that are subsequentlyinactivated) as the vehicle to capture, incorporate and delivermolecules. As an example the M1 Influenza-A matrix protein was used asan illustration, but other viral and/or non-viral components alone or incombination could be envisioned. As a further illustration of theversatility of the invention, HIV or HSV specific antigens are expressedalong with costimulatory molecules to induce immune responses. Incontrast to FIG. 3, the established chronic cell line constituentlyexpresses particles that are continuously released from the said cellline. As a therapeutic, the specificity of the immune response comesfrom the specific inclusion and expression of infectious viralantigens—gp160/120 & gag antigens for HIV; gpB2 & gpD for HSV-2. Thespecific antigens either intact or processed as peptides in majorhistocompatibility (MHC) molecules together with expressed costimulatorymolecules have been shown to elicit corresponding immune responses whenbinding to the appropriate cells either in vitro or in vivo. As adiagnostic, the particles could be used as a preparation for thepurification of specific viral proteins and/or nucleic acids, inaddition to an antigen preparation for detection of immune responsesagainst HIV or HSV in humans by the analysis for the presence ofantibodies against specific viral antigens. Detection could be by ELISA,Western Blot and/or immune fluorescence, but not limited to suchdetection systems. Unlike the previous schematics, all the viralantigens and co-stimulatory molecules contain hybrid constructions madebetween the said molecule and intracellular domain sequences ofInfluenza-A surface proteins—NA refers to the neuraminidase antigen andHA refers to the hemagglutinin antigen, although the addition of thesesequences are not a prerequisite for the inclusion of these saidmolecules into the released particles. In addition, the schematicillustrates the “capturing” of cellular surface molecules that arenative to said host cell, are also present in the final particlepreparation. In this figure, the particles are identified as BC, whichrefer to Biological Carriers. The term biological carriers were coinedto emphasis both the biological nature of the technology and theirability to carry fully expressed molecules.

FIG. 5 is a schematic representation for using virus-like-particlerelease from a continuous particle expressing cell line to incorporatewithin the released particles the cellular receptor (TEM-8) for theProtective Antigen (PA) protein from the bacterium Bacillus anthracis,the etiologic agent of anthrax. As a therapeutic, the releasedparticles—TEM-8 particles—could be used in vitro or in vivo tobio-scavenge PA protein to prevent cellular and tissue damage within thehost either for prophylactic and acute protection. The TEM-8 codingsequence is expressed on the surface of particle budding cells as anin-frame hybrid gene with the Influenza-A hemagglutinin antigenintracellular domain. As a diagnostic, the TEM-8 containing particlescould be used, but not limited to, a detection system within an assay tobind anthrax specific antigen.

FIG. 6 is a schematic representation for using virus-like-particlerelease to incorporate either costimulatory molecules alone orcostimulatory molecules together with a hybrid genetic constructioncontaining the ecto- or extra-cellular domain sequence of the ProtectiveAntigen (PA) protein from the bacterium Bacillus anthracis, theetiologic agent of anthrax. In this figure the particles are made from asingle viral component, the M1 Influenza-A matrix protein, but could bemade from multiple components that are viral, non-viral, or innate toits source cell line. As a therapeutic, the PA antigen either intact orprocessed as peptides in major histocompatibility (MHC) moleculestogether with expressed costimulatory molecules will elicitcorresponding immune responses involving both the humoral andcell-mediated arms of the mammalian immune system, including mechanismspresently known or unknown to eliminate and/or protect by either invitro or in vivo mechanisms the host.

FIG. 7 is a schematic representation for using virus-like-particlerelease to incorporate the Protective Antigen (PA) protein from anthraxinto a particle structure for diagnostic purposes. The PA containingparticles could be used as an anthrax specific antigen for thepurification of this specific viral antigen and/or nucleic acids, inaddition to an antigen preparation for detection of immune responsesagainst anthrax exposure in humans by the analysis for the presence ofantibodies. Detection could be by ELISA, Western Blot and/or immunefluorescence, but are not limited to these systems. The major advantageof incorporating a protein of interest (here the PA protein as anexample) into a particle is for the purification of the antigen usingstandard generic techniques for isolating particles (viral or non-viral)that could be preformed by one skilled in the art for an antigen in theproper orientation, conformation, configuration, multimerization, andpost-translational modifications that include but not limited toglycosylation, polyADPribosylation, and myristylation to improve thefunctional parameters associated with the said molecule(s).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to the use of particles to capture andincorporate recombinant molecules, for the in vitro use of theseparticles, and for the in vivo delivery of these particles within amammalian host as a universal protein delivery system. The inventiondescribes a process and the utility of that process to developtherapeutic entities and diagnostic reagents that can protect, enhance,suppress, alleviate, repair, differentiation, detect, and modulatecellular processes by the delivery of recombinant molecules to influencethese cellular processes in vivo and in vitro.

In the preferred embodiment, the particles used in the invention areproduced in vitro from cells genetically engineered to expressrecombinant molecules onto their cells' surface and geneticallyengineered to produce budding particles that capture and incorporatethese expressed recombinant molecules such that the particles whenharvested contain the recombinant molecules. In accordance with theinvention, the particles are virus-like-particles and as such are notinfectious, but rather serve as biological carriers of expressedrecombinant molecules that are removed from the cells' surface as theparticle is released from the cell. Such particles could be harvestedand then used as recombinant molecules in vitro and in vivo inaccordance with the invention.

The invention provides for the use of the recombinant molecule(s)containing particles to present the relevant molecule(s) to variousbiological processes, for example, as an immunoprophylactic orimmunotherapy to treat cancer, exposure to toxins, infectious diseasesand as an alternative to conventional drug and antibiotic therapies,especially in cases where resistance has developed. Pursuant to thepresent invention, molecules have been expressed and/or induced on thesurface of the continuously expressing particle-producing host cellline, and the released particles are harvested. The recovered particlespresent transduced or endogenously expressed antigen(s) together withco-stimulatory molecule(s) directly to the immune system, or are pickedup by “professional” antigen presenting cells (APCs), such as dendriticcells and macrophages, for presentation to lymphoid cells. The minimumrequirement of an APC for activation of T-lymphocytes are to degradecomplex protein antigens into antigen fragments, to present theseantigen fragments that were bound to MHC molecules present on theparticles by virtue of their presence on the host cell surface andsubsequently captured and incorporated into particles along with therecombinant expressed co-stimulatory molecules, like B7.1 and B7.2.

Other examples of how the invention provides for the use of therecombinant molecule(s) containing particles to present the relevantmolecule(s) to various biological processes can be shown as the use ofthe invention as a bio-scavenging enzyme or receptor, as a cytokine,growth factor, chemo-attractant, and/or a generalized protein, peptide,antibody delivery system in vitro or in vivo to influence variousbiological processes. Pursuant to this embodiment of the invention,molecules have been expressed and/or induced on the surface of thecontinuously expressing particle-producing host cell line, and thereleased particles are harvested. The recovered particles contain thetransduced or endogenously expressed recombinant molecules that wereexpressed on the surface of the continuously expressingparticle-producing host cell line, and the released particles areharvested. The recovered particles contain transduced or endogenouslyexpressed recombinant molecules and these particles could be useddirectly. In vitro, the particles could be used to stimulate biologicalprocesses such as cell growth, expansion, and differentiation—forexample by containing a factor that maintains growth and expansion of aparticular cell type or line without differentiation, or inducingdifferentiation at the expense of growth. This could be the case forembryonic and progenitor stem cells. In addition, the particles could beused in vitro to purify specific molecules in active and native forms oras a reagent for the detection and analysis for the presence ofantibodies or as a control or standard for assays involving the capturedand incorporated recombinant molecule. In vivo, recombinant madeparticles could be used as a generalized protein delivery system capableof delivering proteins, peptides, and antibodies to scavenge toxicmolecules endogenous to human systems or those exogenously introduce—forexample derived from bio-terrorism actions. Also attracting cells to theregion that would assist in the repair process could use in vivo use ofthe invention to deliver necessary factors to influence thedifferentiation and repair of cellular processes involved in cellularand/or tissue regeneration by interacting directly with the repairprocess or by influencing the repair process. In vivo, the particlescould be used to deliver recombinant molecules that influence cellularresponses—for example suppress immune responses during bone marrowtransplantation, influence T-lymphocyte population expansion that mayresult in a favorable response in certain disease conditions and todelivery recombinant molecules to relieve disease symptoms.

Techniques and terms for transduction, sequence isolation, in-framefusions or ligation, gene, recombinant, coding of sequences,intracellular-transmembrane domains, ecto- or extracellular portions ofproteins, genetically-modified, virus-like-particles, virus infection,inactivation of virus particle preparations, viral budding, digestion orrestriction enzyme analyses, in addition to other molecular biologic ormolecular virology techniques and terms that are established and used inthe art are described in standard laboratory manuals and references,such as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 4^(nd) Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.

Pathogens against which the present invention may be applicable in theformation of biological particles containing pathogen antigen(s)include, but are limited to bacteria, parasites, protozoa, fungi, prion,and viruses. Viruses are infectious agents (pathogens) includinghepatitis A, hepatitis B, hepatitis C, herpes simplex viruses, varicellazoster, Epstein-Barr virus, cytomegalovirus, human herpesvirus-6, -7,-8, HIV-1, HIV-2, HTLV-1, HTLV-2, Rubella, Rubeola, Influenza,Rotavirus, West Nile, Dengue and other emerging flaviviruses. Due tobio-terrorism, Category-A biological diseases as define by the CDC,which include: Bacillus anthracis (anthrax), Clostridium botulinum toxin(botulism), Yersinia pestis (plague), Variola mayor (smallpox),Francisella tularensis (tularemia) and pathogens responsible for viralhemorrhagic fevers are included. Prions are the transmissible pathogenicagents responsible for diseases such as scrapie, bovine spongiformencephalopathy, and associated human diseases. Fungi, protozoa andparasites include Toxoplasma, trypanosomes, babesia, rickettsia,malaria, and enteric pathogens. Bacteria include species of Chlamydia,Helicobacter, Neisseria, Mycobacteria, (especially M. tuberculosi). Thescientific literature identifies 1,415 species of infectious organismknown to be pathogenic to humans, including 217 viruses and prions, 538bacteria and rickettsia, 307 fungi, 66 protozoa and 287 helminthes. Outof these, 868 (61%) are zoonotic, that is, they can be transmittedbetween humans and animals, and 175 pathogenic species are associatedwith diseases considered to be “emerging” are included. Over 100 viruseshave been associated with acute central nervous system infections,causing among other diseases encephalitis and meningitis; Nipah virus inMalaysia and neurovirulent enterovirus (70 strains) that cause severeneurological disease; vector borne disease agents include Japaneseencephalitis, Barmah Forest, Ross River, and Chikungunya viruses; hendravirus, formerly called equine morbillivirus a rabies-related virus,Australian bat lyssavirus, and a virus associated with porcinestillbirths and malformations, Menangle virus. Most emerging viruses arezoonotic and because of the large number of present and emergingpathogens that infect human are zoonotic, veterinary viral-deliveredvaccinology strategies are also encompassed within the scope of theinvention.

Antigens against which the present invention may be applicable in theformation of particles containing recombinant forms include polypeptidesencoded by the pathogen listed above. The multitudes of antigens encodedby these agents that may be expressed include, but are not limited toexternal surface proteins and structure proteins including enzymes,transcription factors, and other cell regulatory proteins. For example,antigens encoded by any genes of the HIV-1 genome including gag, pol,vif vpr, vpu, tat, rev, env, and nef may be all present as either intactantigens or immune dominate peptides. Another example is the pathogenicprion protein (PrPSc) template and endogenous cellular prion protein(PrPC). Proteins include all known and to be discovered gene or nucleicacid containing encoded proteins, cytokines and related molecules suchas interleukins, growth factors, chemokines, adhesion molecules,neurotrophic factors, MMPs/TIMPs, receptors, and developmental proteins.Peptides include any amino acid sequence that could be made and/or foundin nature; expressed as monomers or as oligomeric versions, includingimmune-dominant epitopes. Antibodies include polyclonal and monoclonalderived against any human, mammalian, bacterium, parasite, protozoa,fungi, prion, and/or virus antigen. In addition, tumor antigens areincluded in the scope of this invention. Two types of antigens have beenidentified on tumor cells: Tumor-specific transplantation antigens(TSTAs) that are unique to cancer cells, and tumor-associatedtransplantation antigens (TATAs) that are found on both cancer andnormal cells. Thus, tumor antigens consist of TSTAs, TATAs, and oncogeneproteins. Tumor-specific antigens have been identified on tumors inducedby chemical and physical carcinogens and some virally induced tumors.The antigen(s) can be present within the chronic expressing pathogencontaining cell line that is used as the particle-producing host or aspart of an infectious process, naturally native to the cell, transducedor transfected by biological (viral vectors), chemical (liposomes), ormechanical (electroporation) methods. The pathogen antigen could beexpressed and assembled into the pathogen itself, or associated with adifferent pathogen particle.

The following examples further illustrate experiments that havedemonstrated reduction to practice and utility of selected preferredembodiments of the present invention, although they are in no way alimitation of the teachings or disclosure of the present invention asset forth herein.

EXAMPLE 1 As A Bio-Scavenging Enzyme

Production and formation of hepatic cell line expressing mammalianbutyrlcholinesterase and the incorporation of said molecule intovirus-like-particles and the utility of these particles to prevent ordiminish toxicity in vivo demonstrates the principle of this invention.

Butyrlcholinesterase hydrolyze organophosphates, a major component innerve gas and has been shown to protect cells in vivo from bio-terrorismand drug-induced toxicity. A hepatoma cell line was used to stablyexpress on its cell surface, using murine leukemia virus vectors, thebutyrlcholinesterase gene. The heptoma cell line was either subsequentlyvirally infected or contain one or more components (viral, non-viral, orinnate) that resulted in the continuous budding of particles from thesaid cell line. The harvesting of the particles can be done byultracentrifugation or preferably by selective dehydration andprecipitation (use of polyethylene glycol or similar agents), affinityand/or size dependent chromatography. The particles would be tested forbutyrlcholinesterase biological activity in vitro. Optimaldetoxification activity by butyrlcholinesterase requires the tetramericform of the enzyme that is enhanced by the co-expression of aproline-rich attachment domain as a peptide coding the proline-richattachment domain (PRAD) within the said particle-producing cell.Retention times of recombinant appropriate post-translational modifiedbutyrlcholinesterase could be compared to observed retention times ofpurified butyrlcholinesterase from native serum sources. Various routesof delivery will be explored including intravenous, intraperitoneal,intramuscular via autoinjectors, and pulmonary/intranasal via “puffer”devices at different doses to establish optimal bioavailability andretention times. In addition to biowarfare, the recombinant particlesmay be used to alleviate succinylcholine-induced apnea and to treatcocaine or other drug overdosed individuals.

EXAMPLE 2 As A Bio-Scavenging Receptor

Production and formation of cell lines expressing mammalian TumorNecrosis Factor Receptor II and the incorporation of said molecule intoa virus-like-particle and the utility of these particles in binding,sequestering and elimination from a biological system demonstrates theprinciple of this invention.

This example relates to bio-scavenging ability of the invention toscavenge the cytokine TNF-alpha using particles that incorporated theTNF receptor II molecule. Cell lines would be established expressing thesaid molecule onto their cell surface. The cell line could be eithersubsequently virally infected or contain one or more components (viral,non-viral, or innate) that resulted in the continuous budding ofparticles from the said cell line. The harvesting of the particles canbe done by ultracentrifugation or preferably by selective dehydrationand precipitation (use of polyethylene glycol or similar agents),affinity and/or size dependent chromatography. The particles would betested for their ability to bind and sequester TNF-alpha biologicalactivity in vitro, followed by in vivo testing using anarthritic-induced animal model (BalbC SCIDs). Successful demonstrationin animal models could lead to clinical trials in humans.

EXAMPLE 3 As A Cytokine and/or Growth Factor Involved in StimulatingBiological Processes and/or Cellular/Tissue Repair and/or AttractingSpecific Cell Types to Enhance the Repair Process In Vivo

Production and formation of cell lines expressing mammalian cytokinesand/or growth factors either known or yet to be discovered and theincorporation of said molecule(s) into virus particles by acuteinfection of cells harboring said molecules or virus-like-particles, andthe utility of these particles to influence cellular and/or biologicalproperties, including but not limited to receptor binding demonstratesthe principle of this invention.

This example relates to the incorporation and use as a delivery systemin vivo of molecules required or capable of influencing and/or enhancingcellular and tissue regeneration. Cell lines are established expressingthe genetically modified molecule(s) to be expressed on the said cells'surface using techniques established within the art and theincorporation or association of these molecules with viral or non-viral,infectious or non-infectious particles. These particles could bedelivered to a human or mammalian host by oral, suppository,intravenous, intra-muscular, inter-cranial, inter-peritoneal, ordirectly into organs, capillaries, ducts, or lymphoid system eitheralone or associated with biological or non-biological materials ordevices. An inter-respiratory device, cutaneous and topicalapplications, aerosols, creams, puffers, or on surfaces could beenvisioned for particle delivery systemic or local. Surfaces include,but not limited to, synthetic, non-synthetic, biological, ornonbiological matrixes including autologous, allogeneic, and xenogeneicextracellular matrix materials. Dependent on the biological moleculedelivered, the delivery route could be tested for efficacy in animalmodels followed by clinical trials in human.

EXAMPLE 4 As A Cytokine and/or Growth Factor Involved in StimulatingBiological Processes and/or Cellular/Tissue Repair and/or AttractingSpecific Cell Types to Enhance the Repair Process In Vitro

Production and formation of cell lines expressing mammalian cytokinesand/or growth factors either known or yet to be discovered and theincorporation of said molecule(s) into non-viral, virus orvirus-like-particles and the utility of these particles to influencecellular and/or biological properties in culture in vitro, including butnot limited to receptor binding demonstrates the principle of thisinvention.

This example relates to the incorporation and use as a delivery systemin vitro of molecules required or capable of influencing and/orenhancing cellular processes including cellular differentiation and/orhuman and mammalian oocyte activation and the influence towardsblastocyst formation, embryonic stem cell establishment, and/or theself-renewal and/or differentiation of that stem cell line into specificlineages. Cell lines are established expressing the genetically modifiedmolecule(s) to be expressed on the said cells' surface using techniquesestablished within the art and the incorporation or association of thesemolecules with viral or non-viral, infectious or non-infectiousparticles. These particles could be introduced to the culture fluids ofcells to influence cellular processes.

EXAMPLE 5 As A Protein, Peptide, and/or Antibody in vivo Delivery SystemInvolved in Influencing Biological Processes

Production and formation of cell lines expressing mammalian proteins,peptides, and/or antibodies either known or yet to be discovered and theincorporation of said molecule(s) into non-virus, virus orvirus-like-particles and the utility of these particles to influencebiological properties, including but not limited to therapeutic hostdelivery demonstrates the principle of this invention.

This example relates to the incorporation and use as an in vivo deliverysystem, molecules required or capable of influencing biologicalproperties. Properties could include the incorporation of amino acidsequences for proteins and/or antibodies, or of peptides as monomers ormultimers to influence biological properties by inhibiting orstimulating specific biological events, including but not limited toapoptotic and anti-apoptotic factors. Cell lines are establishedexpressing the genetically modified molecule(s) to be expressed on thesaid cells' surface using techniques established within the art and theincorporation or association of these molecules with viral or non-viral,infectious or non-infectious particles. These particles could bedelivered to a human or mammalian host by oral, suppository,intravenous, intramuscular, inter-cranial, inter-peritoneal, or directlyinto organs, capillaries, ducts, or lymphoid system either alone orassociated with biological or non-biological materials or devices. Aninter-respiratory device, cutaneous and topical applications, aerosols,creams, puffers, or on surfaces could be envisioned for particledelivery systemic or local. Surfaces include, but not limited to,synthetic, non-synthetic, biological, or non-biological matrixesincluding autologous, allogeneic, and xenogeneic extracellular matrixmaterials. Dependent on the biological molecule delivered, the deliveryroute could be tested for efficacy in animal models followed by clinicaltrials in human.

EXAMPLE 6 Establishing a Universal Particle Expressing Cell Line forDelivery of any Recombinant Protein, Peptide, and/or Antibody forTherapeutic and/or Diagnostic Purposes

The principle of this invention could be further demonstrated by in vivoexperiments in mice, non-human primates, and ultimately clinicaltrials/treatments in humans.

In the present invention, a virus-based protein delivery system can beengineered to serve as a potential vaccine candidate against infectiousdiseases and cancer. We believe that this system can serve as a generaldelivery system in vivo for any recombinant protein, peptide, and/orantibody based therapeutic, opening up the potential to any and alldisease conditions. To insure safety we have be inactivating and therebydestroying infectivity of infectious virus particles. As a furtherprecaution, we are using virus-like-particles that are innately devoidof the ability to infect cells. The present example uses influenza virusmatrix protein as our core protein to induce a budding process toproduce virus-like-particles, but other single virus components orcombination of components could be envisioned.

The Influenza matrix protein similar to the matrix proteins ofretroviruses, vesicular stomatitis virus, and human parainfluenza virustype 1 has intrinsic budding activity, and when expressed alone, willbud particles into the culture supernatant. We expect these buddingparticles to “capture” cell surface expressed recombinant proteins.Although the interactions between the internal viral component and thecytoplasmic tail of external viral proteins are not an absoluterequirement for particle formation, chimeric constructions ofhemagglutinin—HA and neuramimidase—NA intracellular-transmembranedomains fused in-frame with heterologous extracellular portions ofproteins could further enhance incorporation of recombinant moleculeswithin the released particles. Since the FDA already approves liveattenuated influenza virus vaccines for human administration, using thematrix proteins from influenza virus as our universal particle producingcell line should offer a safe and efficacious method to deliverrecombinant proteins, peptides, and/or antibodies-based therapeutic tohumans.

To establish a M1 Influenza-A matrix protein expressing cell line, RNAwould be isolated from Influenza-A infected MDCK cells, reversedtranscribed, PCR amplified, and the full length M1 matrix protein clonedinto a PCR cloning vector. Oligonucleotides will be synthesizedencompassing the 5′ and 3′ ends of the 759 nucleotide M1 matrix protein(GenBank Accession AF222823) and the cloned fragment will be synthesizedto confirm the accuracy of the amplified fragment. The M1 gene will betransferred into a truncated HIV-LTR clone, pJM167, containing a minimaltat-inducible promoter. Past experience has demonstrated thetat-inducible promoter, in the presence of tat (pJM310) to result inhigh protein expression driven by the continuous transcriptionalactivation by tat binding to the TAR element within the HIV-LTR. Oncethe M1 gene fragment is cloned behind the truncated LTR, this plasmidwill be co-transfected with pJM310 into a series of T- and B-lymphocyticcell lines (Suptl, Hut78, Raji, Molt-3) by clectroporation (to gain thehighest number of integrated gene copies) of the cells in the presenceof media containing the plasmids. The cell lines will be initiallyscreened by RT-PCR for M1 matrix protein expression and the line withthe highest signal will be single cell cloned. Evidence of vesicularparticles in clarified concentrated supernatants of cultured cells willbe confirmed by negative staining electron microscopy particle counts.

EXAMPLE 7 As An Immune Regulatory

Production and formation of cell lines expressing mammalian proteins,peptides, and/or antibodies either known or yet to be discovered and theincorporation of said molecule(s) into non-virus, virus orvirus-like-particles and the utility of these particles to influenceimmune responses, including but not limited to therapeutic host deliverydemonstrates the principle of this invention.

This example relates to the incorporation of immune modulator moleculesinto cell lines that express particles that capture and incorporatessaid molecules. These immune molecules could include one or more of thefollowing proteins, but are not limited to these molecules—B7.1, B7.2,CTLA-4, OXA40, 4-IBB, CD27—that are involved in the activation orsuppression of immune responses. In addition to these molecules in somesituation, specific antigens to infectious disease agents, cancer,and/or autoimmune diseases would be included into the release particlesby their inclusion on to the host cells' surface. In addition to theincorporation of immune and antigen molecules that were exogenouslyexpressed on the cells' surface by standard molecular biologicaltechniques, native cellular expressed molecules are expected to beco-incorporated into the released particles. These molecules wouldinclude processed peptides from the exogenously expressed antigenswithin the groove of MHC class I and class II, plus CD1 molecules. Theprocessed peptides and glycolipids associated with MHC and CD1molecules, respectively, would stimulate immune responses by binding tothe CD3 molecule and the T-cell receptors of appropriate cells, whilethe immune modulator molecules will interact through there respectiveligands or receptors. Although the mechanism of these approaches mightbe induction or repression of immune responses through the humoral andcell-mediated arms of the immune system, other mechanisms may beimplored to affect immune modulation that may involve but not limited tothe expression of cellular factors that influence immune responses. Oneexample is the expression of a CD8+ cell factor that can inhibit HIV-1expression in some HIV-infected individuals.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features herein before set forth.

1. A process for production and formation of a generalized in vivo delivery system that incorporates cell surface expressed recombinant amino acid containing sequences and innate host cell surface cellular molecules within a biologically formed particle, comprising of viral components that bud from host cells that are genetically modified to express cell surface recombinant molecules.
 2. The process of claim 1 wherein said generalized in vivo delivery system deliver amino acid containing sequences that are protein(s), peptide(s), and/or antibodies.
 3. The process of claim 2 wherein the said proteins, peptides, and/or antibodies are expressed on the surface of host cells that produced the budding particles of claim
 1. 4. A method for establishing host cells of claim 1 comprising of viral components that result in budding particles.
 5. The process of genetically modifying said host cells of claim 4 such that the budding particles contain the said proteins, peptides and/or antibodies to be delivered in claim
 2. 6. The process of claim 5 wherein the proteins, peptides, and/or antibodies are passively incorporated into the budding particles by their proximity within the membrane of host cells.
 7. The process of claim 5 wherein the proteins, peptides, and/or antibodies are passively incorporated into the budding particles by inclusion of transmembrane sequences onto the coding sequence of said proteins, peptides, and/or antibodies resulting in membrane expression on host cells.
 8. A method by which the viral components of claim 4 are viral matrix proteins.
 9. A method of claim 8 where the virus is influenza.
 10. The process of claim 7 wherein the proteins, peptides, and/or antibodies are actively incorporation into budding particles by matching viral matrix proteins to the intracellular domains of viral envelope proteins.
 11. The process of claim 1 wherein the budding particles are non-infectious, concentrated, and formulated as a drug for administration to a mammalian recipient.
 12. A process of claim 11 wherein treating a mammalian recipient to provide in said recipient a therapeutic response, comprising administering to a mammalian recipient non-infectious budding particles in an amount effective to be therapeutic.
 13. A process of claim 12 where the non-infectious particles were either previously infectious (containing viral genome) and inactivated or produced from virus-like-particles (missing one or more viral genome components required for infectious virus formation).
 14. A process of claim 12 where the therapeutic response can induce signal transduction pathways by cell surface receptor engagement; induce or inhibit cellular differentiation; stimulate or suppress immune responses; attract or repel cells; prevent host cell and organ toxicity.
 15. A process of claim 14 where the therapeutic response is a vaccine or a prophylactic.
 16. A process of claim 12 where the mammalian recipient is a human.
 17. A process of claim 12 where the mammalian recipient is a non-human primate, canine, feline, or other non-human mammalian species.
 18. A process of claim 1 wherein said host cells are obtained from a cancer recipient.
 19. A process of claim 1 wherein said host cells are obtained from a transplant recipient.
 20. A process of claim 1 wherein said host cells are an established cell line.
 21. A process of claim 1 wherein said host cells are MHC matched.
 22. A process for treating a transplant recipient to reduce in said recipient an immune response to an alloantigen, comprising treating the transplant organ with a therapeutically effective amount of the budding particles of claim 11, wherein either the budding particles are derived from host cells obtained from the donor and/or the budding particles are derived from host cells containing cell surface expressed amino acid containing sequences that code for molecules that can reduce immune response against the alloantigen.
 23. A process for treating a mammalian subject for promoting connective tissue growth, resulting in tissue engineering comprising treating a recipient in need of connective tissue growth by administering a therapeutically effective amount of the budding particles of claim 11, wherein the budding particles are derived from host cells containing incorporated amino acid sequences that code for molecules that can either promote healing or attract endogenous cells that can promote healing.
 24. A process for treating a mammalian subject for prevention of toxicity of biological or chemical agents; comprising treating a recipient exposed or in possibility of exposure by administering a therapeutically effective amount of the budding particles of claim 11, wherein the budding particles are derived from host cells containing incorporated amino acid sequences that code for molecules that can act as a bio-scavenger to either remove or inactivate the toxic substance.
 25. A process for treating a mammalian subject for therapeutic purposes by administering budding particles of claim 11 derived from host cells that display therapeutic moieties as an amino acid sequence where the particles behaved as a drug delivery vehicle.
 26. A process of claim 1 where amino acid containing diagnostic molecules are expressed on the surface of host cells that produce particles; these particles can be used as diagnostic reagents within diagnostic test kits. 